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Transfer of maternal immunity to piglets is involved in early protection againstMycoplasma hyosynoviae infection

Lauritsen, Klara Tølbøl; Hagedorn-Olsen, Tine; Jungersen, Gregers; Riber, Ulla; Stryhn, H.; Friis, N.F.;Lind, Peter; Kristensen, B.Published in:Veterinary Immunology and Immunopathology

Link to article, DOI:10.1016/j.vetimm.2016.12.002

Publication date:2017

Document VersionPeer reviewed version

Link back to DTU Orbit

Citation (APA):Lauritsen, K. T., Hagedorn-Olsen, T., Jungersen, G., Riber, U., Stryhn, H., Friis, N. F., ... Kristensen, B. (2017).Transfer of maternal immunity to piglets is involved in early protection against Mycoplasma hyosynoviaeinfection. Veterinary Immunology and Immunopathology, 183, 22-30.https://doi.org/10.1016/j.vetimm.2016.12.002

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Transfer of maternal immunity to piglets is involved in early 1

protection against Mycoplasma hyosynoviae infection 2

3

4

K. Tølbøll Lauritsen 1,2,*

, T. Hagedorn-Olsen 1, G. Jungersen

1, U. Riber

1, H. Stryhn

5

3, N. F. Friis

1^, P. Lind

1 and B. Kristensen

2 6

7

8

Addresses of authors: 9

10

1 National Veterinary Institute, Technical University of Denmark, Bülowsvej 27, 1870 11

Frederiksberg C, Denmark 12

2 Department of Disease Biology, Faculty of Health and Medical Sciences, University of 13

Copenhagen, Stigbøjlen 4, 1870 Frederiksberg C, Denmark 14

3 Department of Health Management, University of PEI, Charlottetown, Prince Edward Island, 15

Canada. C1A 4P3 Canada 16

17

* Corresponding author: Section for Diagnostic and Scientific Advice, National Veterinary 18

Institute, Technical University of Denmark, Bülowsvej 27, 1870 Frederiksberg C, Denmark 19

E-mail: ktla@vet.dtu.dk, Tel.: +45 35 88 63 72, FAX; +45 35 88 62 30 20

21

Tine Hagedorn-Olsen present address: Dako Denmark A/S, Produktionsvej 42, 2600 Glostrup, 22

Denmark 23

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24

Birte Kristensen present address: Klinik for Veterinær Dermatologi, Skovalleen 31, 2880 25

Bagsværd, Denmark 26

27

^ Deceased. N.F. Friis approved the manuscript before he passed away. 28

29

Abstract 30

Mycoplasma hyosynoviae causes arthritis in pigs older than 12 weeks. The role of 31

colostrum in protection of piglets against M. hyosynoviae infection is not clear. Our 32

objective was therefore to investigate whether transfer of maternal immunity to 33

piglets was involved in early protection against the infection. Experimental infections 34

were carried out in three groups of weaners receiving different levels of 35

M. hyosynoviae-specific colostrum components; Group NC derived from 36

Mycoplasma free sows and possessed no specific immunity to M. hyosynoviae. 37

Group CAb pigs, siblings of the NC group, received colostrum with M. hyosynoviae-38

specific antibodies immediately after birth. Group CCE pigs were born and raised by 39

infected sows and presumably had the full set of colostrally transferred factors, 40

including specific antibodies. When 4½ weeks old, all pigs were inoculated 41

intranasally with M. hyosynoviae. The course of infection was measured through 42

clinical observations of lameness, cultivation of M. hyosynoviae from tonsils, blood 43

and synovial fluid and observation for gross pathological lesions in selected joints. 44

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Specific immune status in the pigs was evaluated through detection of antibodies by 45

immunoblotting and measurement of M. hyosynoviae-specific T-cell proliferation. 46

The latter analysis may possibly indicate that M. hyosynoviae infection induces a T-47

cell response. The CCE piglets were significantly protected against development of 48

lameness and pathology, as well as infection with M. hyosynoviae in tonsils, blood 49

and joints, when compared to the two other groups. Raising the CCE pigs in an 50

infected environment until weaning, with carrier sows as mothers, apparently made 51

them resistant to M. hyosynoviae-arthritis when challenge-infected at 4½ weeks of 52

age. More pigs in group NC had M. hyosynoviae related pathological lesions than in 53

group CAb, a difference that was significant for cubital joints when analysed on joint 54

type level. This finding indicates a partially protective effect of passively transferred 55

M. hyosynoviae-specific colostral antibodies upon development of M. hyosynoviae 56

related pathology. Thus, the level of passive immunity transferred from sow to piglet 57

seems to provide, at least partial, protection against development of arthritis. It cannot 58

be ruled out that the CCE pigs, by growing up in an infected environment, have had 59

the chance to establish an active anti-M. hyosynoviae immune response that 60

complements the maternally transferred immune factors. Evident from this study is 61

that the general absence of M. hyosynoviae arthritis in piglets can be ascribed mainly 62

to their immunological status. 63

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64

Keywords 65

Mycoplasma hyosynoviae, arthritis, colostrum, antibody, pig, lymphocyte 66

proliferation 67

68

Introduction 69

Mycoplasma hyosynoviae infection is a common cause of acute and severe lameness 70

among Danish growing-finishing pigs (Nielsen et al., 2001). Herds with severely 71

affected pigs experience increased use of antibiotics and workload as well as reduced 72

animal welfare (Kobisch and Friis, 1996; Nielsen et al., 2001). The prevalence of M. 73

hyosynoviae in the Danish swine industry has not been investigated thoroughly, 74

however non-published experiences form Danish pig herds indicate that the majority 75

of these are infected. 76

77

M. hyosynoviae is harboured in the tonsils of infected pigs (Ross and Spear, 1973; 78

Friis et al., 1991). This carrier state is primarily established in pigs above ten weeks 79

of age and infection is rarely transmitted from sows to piglets (Hagedorn-Olsen et al., 80

1999a). Via the blood stream the mycoplasmas may spread to the joints (Kobisch and 81

Friis, 1996; Hagedorn-Olsen et al., 1999b) and cause arthritis in pigs above 12 weeks 82

of age (Ross and Duncan, 1970; Hagedorn-Olsen et al., 1999a). A previous 83

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experiment showed that 6-week-old pigs, immunologically naive with respect to M. 84

hyosynoviae, were able to develop acute joint infection within 2 to 13 days after 85

intranasal inoculation with the agent (Lauritsen et al., 2008). This indicated that the 86

absence of M. hyosynoviae related lameness in this age group under field conditions 87

must have another explanation than strictly age related factors. 88

89

Sow colostrum contains antibodies, which the newborn piglets absorb to the 90

circulation through the gut (Frenyo et al., 1981; Klobasa et al., 1981; Rooke and 91

Bland, 2002; Salmon et al., 2009; Bandrick et al., 2011; Nechvatalova et al., 2011), 92

as well as other immunological components such as cells of the immune system, e.g. 93

neutrophils and eosinophils, macrophages and lymphocytes (Evans et al., 1982; 94

Schollenberger et al., 1986a; Schollenberger et al., 1986b; Magnusson et al., 1991, 95

Nechvatalova et al., 2011). Live cells from sow colostrum are transferred across the 96

gut epithelium of the piglet and into the blood/lymphatics (Tuboly et al., 1988; 97

Williams, 1993; Salmon, 2000; Salmon et al., 2009; Nechvatalova et al., 2011) and it 98

has been discussed in several papers whether colostral cells actively could comprise a 99

pool of cellular immunocompetence that can be transferred from sow to the suckling 100

piglet (Salmon, 2000; Wagstrom et al., 2000; Salmon et al, 2009; Nechvatalova et al., 101

2011). The role of colostrum in protection of piglets against M. hyosynoviae infection 102

has so far not been clarified, although the abovementioned results by Lauritsen et al. 103

(2008) may point in the direction of presence of maternally transferred immunity. 104

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Antibodies specific for M. hyosynoviae, presumably originating from colostrum, have 105

been shown to be present in suckling piglets (Blowey, 1993; Hagedorn-Olsen et al., 106

1999a). In the present study the hypothesis was that transfer of maternal immunity to 107

piglets is involved in early protection against Mycoplasma hyosynoviae infection. The 108

possible role of specific M. hyosynoviae antibodies was investigated by developing 109

an experimental colostrum model. One group of piglets were isolated from the sow 110

immediately after birth and fed cell-free colostrum containing significant levels of 111

specific M. hyosynoviae antibodies (Colostrum antibody group - CAb group). 112

Protection of this group after inoculation with M. hyosynoviae was compared to two 113

other groups, one that had suckled infected sows (Complete Colostrum and Exposure 114

group - CCE group) and one that had suckled sows that were immunologically naive 115

to M. hyosynoviae (Naive Colostrum group - NC group). 116

117

Materials and methods 118

Animal material and housing conditions 119

Thirty-two pigs were allocated to three groups, subjected to different regimens of 120

colostrum intake; i) the CAb group received M. hyosynoviae-specific antibodies via 121

colostrum that had been frozen and thawed to destroy live cells, ii) the NC group 122

suckled colostrum without M. hyosynoviae-specific immunity and was therefore 123

immunologically naive, iii) the CCE group received complete colostrum containing 124

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antibodies and cellular components from their M. hyosynoviae infected mothers, and 125

was exposed to infected environment until weaning. The experimental design is 126

illustrated in Fig. 1. All pigs included in the study were cross-breds (offspring from 127

Danish Landrace/Yorkshire sows and Duroc or Hampshire boars). The CAb and NC 128

groups were kept isolated under experimental conditions from birth, whereas the 129

CCE group was transferred to the experimental facilities one week before inoculation 130

(Fig. 1). All pigs of the study were weaned at 3-3½ weeks of age (Fig. 1). The pigs 131

were kept loose in pens with concrete floors and abundant straw-bedding. Fresh water 132

was supplied ad libitum through water nipples, and the pigs were fed factory-made 133

pelleted standard swine feed without addition of any antimicrobials. 134

135

Preparation of the colostrum pool 136

The colostrum artificially fed to the CAb group (Fig. 1) was prepared from colostrum 137

of four sows from a M. hyosynoviae infected herd, but not the same herd which 138

supplied pigs for the CCE group. Within 24 hours after parturition 300-600 ml of 139

colostrum was collected from each sow using the following method; Sows were 140

prepared for colostrum collection by i.v. injection of 20 IU oxytocin (Oxytocin®, Leo 141

Vet) and the udder was washed and disinfected (0.5% chlorhexidin in 70% ethanol). 142

Colostrum was collected by hand stripping into sterile wide mouth glass bottles. After 143

removing 2-4 ml for later cultivation for M. hyosynoviae, 200 mg tiamulin (Tiamutin

144

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R, Novartis) was added per 100 ml colostrum and the colostrum was stored at -20°C. 145

Further, the colostrum was thawed, pooled, filtered through sterile gauze, aliquoted 146

into sterile bottles and stored at -20°C until use. Before being fed to the newborn 147

pigs, the colostrum was thawed in a lukewarm water bath, and the temperature 148

adjusted to 38°C. Further details on the colostrum feeding to piglets are described in 149

Fig. 1. All colostra used were cultivation negative for Mycoplasma spp prior to 150

addition of Tiamulin. 151

152

Inoculation with M. hyosynoviae 153

Between 4 and 4½ weeks of age all pigs were inoculated with a cloned field strain of 154

M. hyosynoviae, Mp927 (titres 107 to 10

8 colour changing units (CCU) per ml). The 155

method used for preparation of inoculum is described by Lauritsen et al. (2008). Pigs 156

were inoculated intranasally into the dorsal meatus, while in dorsal recumbency. 157

Inoculation dose was 1ml in each nostril. 158

159

Mycoplasma cultivation 160

Cultivation for M. hyosynoviae from heparin-stabilized blood samples was performed 161

on post inoculation day (PID) 4, 7, 9, 12 and 15. Tonsillar scrapings, obtained with a 162

sterile blunt steel scraper especially designed for the purpose, were collected two 163

days before inoculation and on PID 4, 7, 9 and 12. Cultivation for M. hyosynoviae in 164

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colostrum was performed in 1:10 serial dilutions to 10-4

in modified Hayflick’s 165

medium (Kobisch and Friis, 1996). All other methods, used in this study for 166

mycoplasma cultivation, including production of inoculation material, have been 167

described by Lauritsen et al. (2008). 168

169

Clinical recordings and post mortem examinations 170

Prior to inoculation all pigs had a normal body condition and did not show any 171

clinical signs of disease. Every day post inoculation, the pigs were observed for 172

clinical signs of lameness and other signs of disease. The pigs were euthanized and 173

autopsied on PID 12, 14 or 16, i.e. in the time period of expected occurrence of the 174

acute infection phase (Kobisch and Friis, 1996; Hagedorn-Olsen et al., 1999c). The 175

date of euthanasia for each pig was determined before inoculation and pigs from each 176

group were evenly represented on the necropsy days. Euthanasia was performed by 177

stunning with a captive bolt pistol followed by exsanguination. At autopsy, six joints 178

per pig were examined for gross pathological lesions since we focused on cubital, 179

stifle and tibiotarsal joints. For each joint the conditions of the synovial fluid and 180

synovial membrane were evaluated by scoring the following seven variables: 181

Synovial fluid colour, volume and transparency, Synovial membrane edema, 182

hyperaemia, hypertrophy and discolouration. In addition the joint cartilages were 183

examined for lesions and discolouration. A pathoanatomical diagnosis was made for 184

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each joint based on the sum of all macroscopic findings recorded for the joint. 185

Synovial fluid was collected aseptically for M. hyosynoviae cultivation from these 186

joints and transferred to a sterile tube containing mycoplasma transport medium 187

(Kobisch and Friis, 1996). The amount of synovial fluid used for cultivation varied 188

depending on the amount obtainable from the joints - from one drop to one ml. The 189

synovial fluid samples were also examined for the presence of M. hyorhinis, 190

M. hyopneumoniae and M. flocculare by cultivation. From each pig, the tonsils were 191

collected for M. hyosynoviae cultivation. 192

The procedures related to animal experimentation had been approved by the Danish 193

Animal Experiments Inspectorate (Licence No. 1999/561-207). 194

195

M. hyosynoviae antigen for lymphocyte proliferation assay and immunoblots 196

Pelleted (1.6 g) M. hyosynoviae species type strain S16 (Ross and Karmon, 1970) 197

resuspended in 10 ml sterile Milli Q water was subject to 10 repeated freeze-thaw 198

cycles and finally centrifugated at 1000 x g, 30 min. The washed pellet was 199

solubilized twice on ice in NP40 lysis buffer (2 % v/v Nonidet P40, 2 mM EDTA, 0.1 200

mM IAA and 1 mM PMSF in PBS), and centrifuged at 20000 x g after which the new 201

pellet was boiled for 5 minutes in 3 ml lysis buffer with addition of 2 % w/v SDS and 202

centrifuged at 20000 x g for 30 min. Free SDS was removed by ultrafiltration through 203

an YM 10 (Amicon) membrane at 4C. The resulting antigen solution was called 204

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Mhyos-antigen. It was aliquoted and stored at -20C. A protein concentration of 3.3 205

mg/ml was measured by the Micro BCA (bicinchoninic acid) Protein Assay (Pierce) 206

using bovine serum albumin as standard. To assure that the antigen exerted no 207

inhibitory effect on cell cultures, an MTT test (Mosmann, 1983) for antigen toxicity 208

and non-specific stimulation was performed. The antigen preparation induced low 209

non-specific activity at 20 g/ml. However, at lower concentrations no non-specific 210

activity was observed and the antigen was non-toxic at all concentrations. 211

212

BrdU lymphocyte proliferation assay 213

Antigen-specific lymphocyte proliferation in response to M. hyosynoviae challenge 214

infection was investigated in blood samples from all pigs two days before inoculation 215

and on PID 7 and 12. Proliferation was measured by flow cytometry, assessing cells 216

that had incorporated the thymidine analog Bromo-deoxy-Uridine (BrdU) in newly 217

synthesized DNA (Riber and Jungersen, 2007). Briefly, peripheral blood 218

mononuclear cells (PBMCs, 3 x 106/ml) in cell culture medium (RPMI 1640 with 219

GlutaMAX™ I, foetal calf serum (10%), penicillin (100 U/ml), streptomycin (100 220

g/ml)) were incubated in 24 well, cell culture plates (Greiner Labortechnik GmbH, 221

Germany): SEB-culture (Staphylococcal enterotoxin B, 5 g/ml, Alexis, Grünberg, 222

Germany), Ag-culture (Mhyos-antigen, 10 g/ml), RPMI-culture (nil-stimulation). 223

Incubation was performed for 5 days at 37C in 5% CO2, the last 18 hours with 224

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addition of 5-Brom-2’-Deoxyuridine (BrdU 60 M, Sigma-Aldrich, St. Louis, MO, 225

USA). 226

Cells were harvested and stained with mAb against swine CD3 (clone PPT3, Yang et 227

al 1996) and secondary R-phycoerythrin conjugated antibody (R0439, DAKO, 228

Denmark). Then cells were fixed with BD-lysis-solution (BD biosciences) and 229

permeabilized with BD-permeabilizing-solution (BD Biosciences) and stained with 230

FITC conjugated Mab against BrdU containing DNAse (BD Biosciences). As control 231

for BrdU staining, cells were incubated with isotype-control antibody (X0927, 232

DAKO, Denmark). Cells were analysed on FACScan by use of CellQuest software 233

(BD Biosciences). 234

20000 gated cells (interpreted as live lymphocytes) were acquired and CD3+BrdU+ 235

double positive cells, i.e. T-cells that have proliferated, were measured (see 236

supplementary material for details). Mhyos-antigen-specific lymphocyte proliferation 237

was calculated as: %CD3+BrdU+ cells (Ag-culture) with subtraction of 238

%CD3+BrdU+ (RPMI-culture). 239

Detection of antibodies 240

Sera from the pigs collected before inoculation (when pigs were 2-3 days, 2 and 4 241

weeks of age) and colostrum samples from sows no. 1-4 were tested by 242

immunoblotting for the presence of specific antibodies against M. hyosynoviae; The 243

Mhyos-antigen was diluted in sample buffer (4 NOVEX NuPAGE Sample Buffer, 244

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San Diego, CA) containing 100mM -mercaptoethanol. For the electrophoresis, 245

NuPAGE 4-12% Bis-Tris gels (NOVEX) were used in running buffer (20 NOVEX 246

NuPAGE MOPS SDS Running Buffer). SeeBlue PreStained Standards (NOVEX) 247

were used as marker. Blotting was performed in NuPAGE Transfer Buffer (NP0006, 248

NOVEX) and membranes were blocked with TBS + 0.5 % Tween-20. Nitrocellulose 249

strips cut from the blots were incubated overnight with either serum or colostrum 250

(dilutions 1:200 or 1:500 in TBS + 0.5 % Tween-20, respectively). HRP-conjugated 251

rabbit anti-swine Ig antiserum (DAKO cat.no. P164, 1:2000 in TBS + 0.5 % Tween-252

20) was used as secondary antibody. Between each step, the strips were washed with 253

TBS + 0.5 % Tween-20. Finally the strips were washed for 10 minutes in 50 mM 254

sodiumacetate and the protein bands were developed in dioctyl sodium sulfasuccinate 255

(DSS)/tetramethylbenzidine-solution for 1 to 15 minutes. The strips were then 256

washed in a DSS-solution for maximum 15 minutes and finally dried, after which the 257

presence of M. hyosynoviae-specific bands was evaluated. Defining the bands that 258

were specific for M. hyosynoviae was performed by comparing Western blot band 259

patterns obtained with serum of experimentally infected pigs (sera supplied by Dr. 260

Niels Filskov Friis). Recognition of two bands at level with the 191kDa size marker 261

(Fig. 2) was consistent in all expectedly positive pigs and was absent in naive pigs 262

(data not shown) as well as in pigs infected with other swine specific mycoplasmas. 263

These two bands were used for differentiating between seropositive and seronegative 264

pigs (Fig. 2). 265

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266

Additionally the total Ig content in serum from 2 to 5 days old pigs was measured in a 267

non-competitive direct ELISA, using plates coated with rabbit anti-swine Ig 268

antiserum (DAKO Z0139). As secondary antibody HRP-conjugated, rabbit anti-269

swine Ig antiserum (DAKO P0164) was used. Ig concentration in serum samples was 270

calculated from a standard curve of two-fold dilutions of normal swine Ig fraction 20 271

mg/ml (DAKO X0906) (start dilution 3.125 ng/ml). 272

273

Statistical analysis 274

Multiple measurements on the same pig were, whenever possible without substantial 275

loss of information, aggregated into a single measure reflecting the overall status of 276

the pig. This approach facilitates the biological interpretation of the results and avoids 277

complex modelling of discrete repeated measures outcomes (Diggle et al., 2002). 278

Cultivation of blood samples and recordings of clinical signs of lameness during the 279

period from challenge to autopsy were interpreted in parallel, that is, the pig was 280

considered a positive reactor if at least one recording was positive. Cultivation of 281

tonsillar samples at autopsy were positive for almost all pigs, and an additional 282

analysis was therefore carried out for records of whether pigs had only positive 283

samples (negative interpretation in parallel). Autopsy results (synovial fluid 284

cultivations, pathological findings) for multiple joints were both interpreted in 285

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parallel across joints and analysed separately for cubital, stifle and tibiotarsal joints 286

because M. hyosynoviae arthritis seems to be more frequently observed in some joints 287

than in others (Ross, 1973). As the majority of joints in two of the groups showed no 288

pathological lesions, a presence/absence recording of lesions was preferred over 289

using scores of the individual arthritis severity. 290

291

The statistical procedure used to compare the groups with respect to dichotomous 292

outcomes at the pig level was a logistic regression controlling for confounding of 293

experiment (1 or 2), litter and day of measurement (autopsy recordings only) by fixed 294

effects. The potential confounders were omitted when statistically clearly non-295

significant (p>0.10) and without any substantial confounding effect (less than 20% 296

change in odds-ratio (Dohoo et al., 2009)). The odds-ratio expresses roughly the 297

factor by which the occurrence of M. hyosynoviae related findings was higher in one 298

group (e.g., NC) relative to another group (e.g., CAb). The effectiveness of 299

controlling for experiment was confirmed by additional Mantel-Haenszel analyses 300

and Generalised Estimating Equation (GEE) logistic regression (Davis, 2002) with an 301

exchangeable correlation structure. Analyses for the NC and CAb groups of synovial 302

fluid and arthritis outcomes at multiple joints used a similar GEE logistic regression 303

to account for two joints (of each type) being measured in each pig. In addition to the 304

logistic regression analyses, Fisher's exact test was used for outcomes that were 305

constant within at least one group. As described by Greenland et al. (2016), we 306

- 16 -

interpreted the p-values as continuous measures that express the compatibility 307

between the data and the statistical model used. The significance level was set at 308

p<0.05. Some findings close to statistical significance were noted as such because of 309

their potential interest, but they were not treated as significant results in the 310

discussion and conclusion. All analyses were carried out by the statistical software 311

SAS, version 9. 312

313

T-cell proliferation was compared among the three groups by the non-parametric 314

Kruskal-Wallis test, supplemented with comparisons between selected pairs of 315

groups by the non-parametric Mann-Whitney test, using GraphPad Prism version 316

5.02, GraphPad Software, San Diego California USA, www.graphpad.com. 317

318

Results 319

Clinical signs 320

Among the pigs in the NC and CAb groups there were some registrations of lameness 321

post challenge (Table 1). The CCE group had no lameness registrations and was 322

found to differ significantly from the NC group (p=0.007) (Table 1). The difference 323

between group NC and group CAb was not statistically significant (p=0.22). 324

325

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Gross pathological findings 326

The observed gross pathological lesions were of varying severity ranging from slight 327

serous arthritis (slightly increased volume of synovial fluid that might be discoloured 328

and turbid. In synovial membrane: mild edema, hyperaemia and/or discolouration), 329

hyperplastic arthritis (varying degree of increase in both synovial fluid volume, 330

discolouration and turbidity. The synovial membrane showed pronounced 331

hyperplasia, sometimes edema, and quite often hyperaemia and discolouration) or 332

serofibrinous arthritis (significantly increased synovial fluid volume with some 333

discolouration and pronounced turbidity, the synovial membrane had marked edema, 334

and some discolouration). Several pigs in groups NC and CAb had M. hyosynoviae 335

related pathological lesions, a marked difference to group CCE with no such findings 336

(Table 1). For all joint types, more pigs in group NC had pathological lesions than in 337

group CAb. The difference was statistically significant for cubital joints (odds-338

ratio=29, p=0.002) and close to significant for stifle joints (odds-ratio=6.4, p=0.095) 339

(Table 2). For tibiotarsal joints, the day of autopsy had a significant effect; day 16 340

post challenge had a higher occurrence of pathological lesions than preceding days 341

(odds-ratio=14, p=0.003). 342

343

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Synovial fluid cultivations 344

Cultures of synovial fluid from the joints from all pigs in group CCE were negative 345

for M. hyosynoviae while M. hyosynoviae was demonstrated in joints from the 346

majority of pigs in both groups NC and CAb (Table 1). For both cubital, stifle and 347

tibiotarsal joints, more pigs had positive cultures in group NC than in group CAb. 348

These differences were not statistically significant (Table 2), even if the comparison 349

between NC and CAb groups for stifle joints was close to significant (odds-ratio 350

>1000, p=0.086). M. hyorhinis, M. hyopneumoniae and M. flocculare were not 351

isolated from any of the synovial fluid samples. 352

353

Cultivation of M. hyosynoviae from tonsils and blood 354

Prior to inoculation tonsillar scrapings from all pigs were M. hyosynoviae negative 355

except from one pig in group CCE. Contrary to this, all but one pig from the CCE 356

group, were tonsil carriers at autopsy (Table 1). While almost all pigs in groups NC 357

and CAb were positive on all repeated samplings post inoculation, the pigs in group 358

CCE had any number between 0 and 5 (maximum) positive samplings. One 359

interpretation of this pattern is that pigs in the CCE group tended to develop a carrier 360

state later than pigs of the other groups. This was reflected in a significant difference 361

between the CCE group and the two other groups but no significant difference 362

- 19 -

between the latter groups, when analysing positive cultivations on PID 4 (results not 363

shown). 364

365

Group CCE pigs had no positive blood samples during the experiment while all pigs 366

in groups NC and CAb experienced a haematogenous phase (Table 1). The majority 367

of pigs in the NC and CAb groups had the same pattern of cultivation positive blood 368

samples, with the first three samples (PID 4, 7 and 9) being culture positive. 369

370

Lymphocyte proliferation 371

The T-cell proliferation assay was implemented in a previous experiment including 372

four M. hyosynoviae inoculated pigs (13 weeks old) and one non-inoculated control 373

pig (unpublished data, pigs described by Lauritsen et al. (2008)). Signs of specific 374

lymphocyte proliferation against Mhyos-antigen were found on PID 11 in the four 375

inoculated pigs (CD3+BrdU

+ cells: 1.4%; 2.2%; 7.2%; 7.6%), but not in the control 376

pig (0.81%). No differences in level of proliferation were observed between Ag-377

cultures with either 2 g/ml or 10 g/ml of Mhyos-antigen. 378

In the present study %CD3+BrdU

+ in RPMI-cultures varied quite a lot, and 379

particularly on PID 7 high %CD3+BrdU

+ was measured in RPMI-cultures in some 380

pigs from all groups (Mean: 4.6%, Range 1.0-15.6%), which in some cases could be 381

related to positive cultivation of M. hyosynoviae in blood samples. Contrary to this, 382

- 20 -

most samples from PID 12 had a %CD3+BrdU

+ in RPMI-cultures around 1% (Mean: 383

1.4%, Range 0.4-3.9%), and only one pig had a M. hyosynoviae cultivation-positive 384

blood sample on this day. Therefore, comparison of antigen-specific proliferative 385

response in the three treatment groups was only performed at PID 12. As shown in 386

Fig. 3, there was a large variation in degree of proliferation on pig level seen as an 387

individual variation within the groups, concerning the antigen-specific proliferative 388

response that we have measured on PID 12. We found no significant differences 389

between the three treatment groups (P=0.35, Kruskal-Wallis test). The medians of the 390

CAb and CCE groups (i.e. the groups appearing most different in Fig. 3) were not 391

statistically different (P=0.16, Mann-Whitney test). Likewise, the difference in the 392

medians of the NC and CCE groups was statistically non-significant (P=0.37) on PID 393

12. 394

395

Antibody responses 396

Evaluating the serum antibody profiles of pigs in the three groups by immunoblotting 397

revealed that the NC group possessed no bands specific for M. hyosynoviae at any 398

time prior to inoculation (Fig. 2). Contrary to this, immunoblots from all pigs of 399

groups CAb and CCE revealed bands specific for M. hyosynoviae prior to 400

inoculation, but the general band patterns of these two groups differed from one to 401

another (Fig. 2). In accordance with the findings in the pigs, colostrum from the 402

- 21 -

M. hyosynoviae immunologically naive sows (no. 1 and 2) had no M. hyosynoviae 403

specific bands whereas specific bands were found in colostrum of sows no. 3 and 4 404

from the infected herds and in the colostrum pool used for feeding the piglets. The 405

average total immunoglobulin content in serum from 2- to 5-day-old pigs, measured 406

in ELISA, were for the CAb group 16 mg/ml, the NC group 30 mg/ml and the CCE 407

group 32 mg/ml. 408

409

Discussion and conclusion 410

The result of the immunoblottings confirmed that the specific immune statuses of the 411

pigs were the following on the day of inoculation with M. hyosynoviae; i) The pigs 412

from sows that were immunologically naive (group NC) possessed no specific 413

immunity against the agent. ii) The colostrum treated pigs (group CAb pigs) 414

possessed specific antibodies as a consequence of the artificial colostrum 415

administration. iii) The pigs from infected sows (group CCE) had received specific 416

antibodies, and presumably the full set of maternally transferred factors from 417

colostrum of their infected dams. 418

419

The course of infection after inoculation in the CCE group differed significantly from 420

that of the two other groups for several parameters measured; no signs of clinical 421

arthritis or gross pathological findings was found in the CCE group, and cultivation 422

of M. hyosynoviae from tonsils, blood and joints of these pigs was significantly 423

- 22 -

reduced compared to the other groups. With respect to immunological status and 424

M. hyosynoviae-infection status, the CCE group represent the population of newly 425

weaned pigs in most Danish herds. They possess M. hyosynoviae-specific antibodies 426

just as it has been shown to be the case for piglets from infected sows in Danish herds 427

(Hagedorn-Olsen et al., 1999a). Also the limited effect of the experimental infection 428

in the CCE pigs is in accordance with the observation that clinical M. hyosynoviae 429

arthritis does not affect pigs below 30-40 kg in infected herds (Kobisch and Friis, 430

1996). Raising the CCE pigs in an infected environment until weaning, with carrier 431

sows as mothers, apparently made these pigs resistant to M. hyosynoviae-arthritis, 432

when challenge infected at 4 weeks of age. However observational studies performed 433

in infected herds indicate that this, probably maternally-derived, protection is of 434

limited duration and that pigs become susceptible to M. hyosynoviae infection and are 435

at risk of developing M. hyosynoviae-related arthritis later in life (Ross and Spear, 436

1973; Hagedorn-Olsen et al., 1999a). 437

438

Previous intranasal inoculation experiments with M. hyosynoviae, performed in M. 439

hyosynoviae-free pigs, have shown that 13 to 17-week-olds experienced a 440

generalisation phase from PID 2, with no detectable mycoplasmas in blood after PID 441

9 (Hagedorn-Olsen et al., 1999b) and that 6-week-old pigs, immunologically naive 442

with respect to M. hyosynoviae, primarily had bacteremia at PID 4, 6 and 8 (Lauritsen 443

et al., 2008). This is in accordance with the findings in groups NC and CAb, where 444

- 23 -

positive blood cultivations were predominantly seen on PID 4, 7 and 9. The lack of 445

positive blood cultivations in group CCE pigs during the experiment, i.e. no 446

detectable generalisation from PID 4 and forth, indicates a very short period of, or 447

maybe a total lack of haematogenous spread of M. hyosynoviae in this group. 448

449

The experimental infection resulted in occurrence of clinical M. hyosynoviae-arthritis 450

both in pigs of group NC and group CAb. Also gross pathological findings 451

compatible with M. hyosynoviae-infection, bacteremia and early tonsillar 452

colonization were observed in these groups. The presence of an ongoing disease 453

condition in the pigs of the NC group was also reflected by a drop in the negative 454

acute phase protein, transthyretin, 7 days after infection (Heegaard et al. 2011). The 455

higher frequency of M. hyosynoviae related pathological lesions in cubital joints upon 456

challenge infection in group NC, as compared to group CAb, may indicate a partially 457

protective effect of the intake of M. hyosynoviae-specific colostral antibodies upon 458

development of M. hyosynoviae related pathology. The average total immunoglobulin 459

content in serum from 2- to 5-day-old pigs, as measured in ELISA, showed that the 460

colostrum treated pigs possessed the lowest concentration of total IgG in serum. 461

Therefore the observed, partially protective, effect of the colostrum treatment on 462

development of arthritis can be ascribed to M. hyosynoviae specific antibodies, and 463

not to a generally higher level of non-specific maternally transferred 464

immunoglobulins. 465

- 24 -

466

The role of specific antibodies in M. hyosynoviae infection is not clear, and could 467

include elimination of the mycoplasmas (neutralisation, opsonisation, complement 468

activation) as well as creation of pathology during infection (e.g. deposition of 469

immune complexes). The partially protective effect of M. hyosynoviae specific 470

antibodies on development of arthritis found in this study is contradictory to the 471

findings in pigs above 10 weeks made by Blowey (1993). He found that the 472

appearance of arthritis in gilts was independent of the level of specific antibodies in 473

serum, indicating that the antibody level was of lesser importance for protection 474

against the disease. Also conflicting herd observations, describing antibody levels in 475

relation to bacteremia with M. hyosynoviae, have been reported; Hagedorn-Olsen et 476

al. (1999a) found cases of pigs that had raised a specific serological response against 477

M. hyosynoviae, after which they developed a generalisation phase with the agent. 478

Contrary to this, Nielsen et al. reported that 3 to 5-month-old pigs with bacteremia 479

had a lower level of M. hyosynoviae specific antibodies, than pigs with no 480

demonstrable bacteremia (Nielsen et al., 2005). The general ability of mycoplasmas 481

to vary their surface antigens to evade the host immune response (Razin et al., 1998) 482

may make antibody-mediated elimination difficult to obtain and could be an 483

explanation for the above mentioned ambiguous effect of antibodies. Thus the 484

importance of antibodies in protection against M. hyosynoviae arthritis is equivocal. 485

486

- 25 -

It has not previously been investigated whether M. hyosynoviae infection induces a 487

specific T-cell response. In this study we found a marked variation between 488

individual pigs concerning specific T-cell responses post inoculation. However, T-489

cell responses are in general widely fluctuating over time and between individuals, so 490

we would not expect all pigs to mount a synchronised and similar specific T-cell 491

response post inoculation. The high percentage of specific proliferation in response to 492

M. hyosynoviae antigen found in some pigs on PID 12 indicates that M. hyosynoviae 493

infection has the potential of inducing an antigen-specific T-cell response. The 494

involvement of this response in protection against the infection remains uncertain, 495

however, it might be that a cell-mediated immune response participate in protection. 496

We found in this study no statistically significant difference between the three 497

treatment groups, with respect to percentages of M. hyosynoviae antigen-specific T-498

cell proliferation. 499

500

The design of the study did not take differences in M. hyosynoviae strains into 501

account. Ross et al. (1978) demonstrated different M. hyosynoviae strains by 502

serological and electrophoretic methods, and Kokotovic et al. (2002a; 2002b) found a 503

pronounced genetic diversity in chromosomal fingerprints performed on Danish herd 504

isolates of M. hyosynoviae. In our study several strains could be involved; 1) the 505

inoculation material, 2) the strain(s) present in the herd that supplied the colostrum 506

batch, and 3) the strain(s) present in the origin herd of the group CCE pigs. We found 507

- 26 -

differences in band pattern between immunoblots of pigs in the CAb and the CCE 508

groups. However, the immunoblots reveal many bands of which we do not know how 509

many are M. hyosynoviae-specific. Thus we do not know whether we worked with at 510

homo- or heterologous system, or a mixture of the both, in this challenge experiment. 511

Designing the study in a way so that all infected sows were infected with a strain 512

similar to that of the inoculation material was neither economically nor practically 513

feasible. 514

515

Based on the results of our study, we conclude that in contrast to immunologically 516

naive piglets, piglets that have been raised in an infected environment and have 517

suckled infected sows are protected against early infection with M. hyosynoviae and 518

development of arthritis when challenge infected at 4½ weeks of age. We found 519

indications that this protection is related to the level of passive immunity because M. 520

hyosynoviae-specific maternally transferred antibodies provided at least partial 521

protection against development of arthritis in otherwise immunologically naive pigs. 522

For comparison, a similar protective effect of colostral antibodies against another 523

mycoplasma, Mycoplasma hyopneumoniae, has been described by Rautiainen & 524

Wallgren (2001), Wallgren et al. (1998) and Siblia et al. (2008). However the marked 525

difference between groups CAb and CCE indicates that something more than 526

maternally derived antibodies contributes to the protection. It cannot be ruled out that 527

the CCE pigs, by growing up in an infected environment, have had the chance to 528

- 27 -

establish an active immune response against the agent that complements the 529

maternally transferred immune factors, although only one of the 13 pigs in this group 530

was cultivation positive in the tonsils prior to inoculation. Alternatively the observed 531

high level of resistance to the challenge infection in the pigs that had suckled infected 532

sows could be due to the uptake of cellular components, e.g. primed lymphocytes, 533

from the fresh sow colostrum. By using a model antigen, Nechvatalova et al. (2011), 534

showed that sows via colostrum were able to transfer antigen-specific lymphocytes to 535

the mesenteric lymph nodes and blood stream of their offspring. Likewise Hlavova et 536

al. (2014), found that colostrum contained high numbers of antigen-experienced 537

lymphocytes with a central/effector memory function, that might play a role as 538

passive immunity in offspring, besides having a local mucosal immune defence effect 539

in mammae of the sow. Oh et al. (2012) demonstrated passive transfer of maternally 540

derived PCV-2-specific cellular immune response to piglets from colostrum, by 541

measuring intradermal delayed type hypersensitivity responses and specific blood 542

lymphocyte proliferation in piglets from vaccinated sows. For the other swine 543

pathogenic mycoplasma, Mycoplasma hyopneumoniae, Bandrick et al. (2008, 2014) 544

have described the transfer of functional antigen-specific T-cells from sows to their 545

offspring that leaves the newborn piglet able to mount an antigen-specific secondary 546

immune response. They further stated that this transfer of Mycoplasma 547

hyopneumoniae-specific cellular immunity is dependent on the piglet suckling its 548

- 28 -

biological mother sow (Bandrick et al., 2011). Something similar could take place 549

between M. hyosynoviae infected sows and their offspring too. 550

551

We have earlier induced clinical M. hyosynoviae arthritis experimentally in 6-week-552

old piglets from a mycoplasma free herd (Lauritsen et al., 2008), and the findings in 553

the NC group confirms the absence of a strictly age related insusceptibility to M. 554

hyosynoviae arthritis in young pigs. Regardless of whether it is primarily acquired 555

antibodies (and cells) or also an active immune response that causes the high level of 556

protection in the CCE group, it is evident that the general absence of arthritis in herd 557

piglets can be ascribed mainly to their immunological status with respect to M. 558

hyosynoviae. Protective immunity against the infection is apparently achievable in 559

piglets, a fact that is of importance e.g. when considering development of an effective 560

immune prophylaxis. 561

562

Conflict of interest 563

The authors declare that they have no competing interests. 564

565

Acknowledgements 566

The project was funded by The Research Centre for the Management of Animal 567

Production and Health (CEPROS), Denmark (project no. CEP-SVS97-7). Hans 568

- 29 -

Skåning and staff in the animal facility at National Veterinary Institute are thanked 569

for their great help. Also laboratory technicians Ulla Amtoft, Parvin Faghan, Jesper 570

Juehl Hansen and Jan Lauritsen are thanked for assistance with Mycoplasma 571

cultivation, cell culture, antigen production, and serological testing. Associate 572

Professor N. C. Nielsen from the University of Copenhagen is thanked for invaluable 573

input on the experimental planning and design. John Lund and his staff from the 574

experimental herd related to Faculty of Health and Medical Sciences, University of 575

Copenhagen, are thanked for supplying colostrum. 576

577

References 578

579

Bandrick, M., Pieters, M., Pijoan, C., Molitor T.W., 2008. Passive transfer of maternal Mycoplasma 580

hyopneumoniae-specific cellular immunity to piglets. Clinical and Vaccine Immunology 15, 540-581

543. 582

Bandrick M., Pieters M., Pijoan C., Baidoo S.K., Molitor T.W., 2011. Effect of cross-fostering on 583

transfer of maternal immunity to Mycoplasma hyopneumoniae to piglets. Vet. Rec. 168, 100. 584

Bandrick M., Ariza-Nieto C., Baidoo S.K., Molitor T.W., 2014. Colostral antibody-mediated and 585

cell-mediated immunity contributes to innate and antigen-specific immunity in piglets. Dev. Comp. 586

Immunol. 43, 114-20. 587

Blowey, R.W., 1993. Mycoplasma hyosynoviae arthritis. Pig Veterinary Journal 30, 72-76. 588

- 30 -

Davis, C.S., 2002. Statistical Methods for the Analysis of Repeated Measurements. Springer, New 589

York, USA. 590

Diggle, P.J., Heagerty, P., Liang, K.-Y., Zeger, S.L., 2002. Analysis of Longitudinal Data. Oxford 591

University Press, Oxford, UK. 400 pp. 592

Dohoo, I., Martin, W., Stryhn, H., 2009. Veterinary Epidemiologic Research. AVC-Inc, 593

Charlottetown, Canada. 865 pp. 594

Evans, P.A., Newby, T.J., Stokes, C.R., Bourne., F.J., 1982. A study of cells in the mammary 595

secretions of sows. Vet. Immunol. Immunopathol. 3, 515-527. 596

Frenyo, V.L., Pethes, G., Antal, T., Szabo, I., 1981. Changes in colostral and serum IgG content in 597

swine in relation to time. Vet. Res. Commun. 4, 275-282. 598

Friis, N.F., Ahrens, P., Larsen, H., 1991. Mycoplasma hyosynoviae isolation from the upper 599

respiratory tract and tonsils of pigs. Acta Vet. Scand. 32, 425-429. 600

Greenland, S., Senn, S.J., Rothman, K.J., Carlin, J.B., Poole, C., Goodman, S.N., Altman, D.G., 601

2016. Statistical tests, P values, confidence intervals, and power: a guide to misinterpretations. Eur J 602

Epidemiol, 31, 337-50 603

Hagedorn-Olsen, T., Nielsen, N.C., Friis, N.F., Nielsen. J., 1999a. Progression of Mycoplasma 604

hyosynoviae infection in three pig herds. Development of tonsillar carrier state, arthritis and 605

antibodies in serum and synovial fluid in pigs from birth to slaughter. Zentralbl. Veterinarmed. A 606

46, 555-564. 607

- 31 -

Hagedorn-Olsen, T., Nielsen, N.C., Friis, N.F., 1999b. Induction of arthritis with Mycoplasma 608

hyosynoviae in pigs: clinical response and re-isolation of the organism from body fluids and organs. 609

Zentralbl. Veterinarmed. A 46, 317-325. 610

Hagedorn-Olsen, T., Basse, A., Jensen, T.K., Nielsen, N.C., 1999c. Gross and histopathological 611

findings in synovial membranes of pigs with experimentally induced Mycoplasma hyosynoviae 612

arthritis. APMIS 107, 201-210. 613

Heegaard, P.M., Stockmarr, A., Piñeiro, M., Carpintero, R., Lampreave, F., Campbell, F.M., 614

Eckersall, P.D., Toussaint, M.J., Gruys, E., Sorensen, N.S., 2011. Optimal combinations of acute 615

phase proteins for detecting infectious disease in pigs. Vet Res. 42:50. 616

Hlavova, K., Stepanova, H., Faldyna, M., 2014. The phenotype and activation status of T and NK 617

cells in porcine colostrum suggest these are central/effector memory cells. Vet J. 202, 477-82. 618

Klobasa, F., Werhahn, E., Butler, J.E., 1981. Regulation of humoral immunity in the piglet by 619

immunoglobulin of maternal origin. Res. Vet. Sci. 31, 195-206. 620

Kobisch, M., Friis., N.F., 1996. Swine mycoplasmoses. Rev. Sci. Tech. 15, 1569-1605. 621

Kokotovic, B., Friis, N.F., Ahrens, P., 2002a. Characterization of Mycoplasma hyosynoviae strains 622

by amplified fragment length polymorphism analysis, pulsed-field gel electrophoresis and 16S 623

ribosomal DNA sequencing. J. Vet. Med. B 49, 245-252. 624

Kokotovic, B., Friis, N.F., Nielsen, E.O., Ahrens. P., 2002b. Genomic diversity among Danish field 625

strains of Mycoplasma hyosynoviae assessed by amplified fragment length polymorphism analysis. 626

Vet. Microbiol. 85, 221-231. 627

- 32 -

Lauritsen, K.T., Hagedorn-Olsen, T., Friis, N.F., Lind, P., Jungersen, G., 2008. Absence of strictly 628

age-related resistance to Mycoplasma hyosynoviae infection in 6-week-old pigs. Veterinary 629

Microbiology 130, 385-390. 630

Magnusson, U., Rodriguez-Martinez, H., Einarsson, S., 1991. A simple, rapid method for 631

differential cell counts in porcine mammary secretions. Vet. Rec. 129, 485-490. 632

Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to 633

proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63. 634

Nechvatalova, K., Kudlackova, H., Leva, L., Babickova, K., Faldyna, M., 2011. Transfer of 635

humoral and cell-mediated immunity via colostrum in pigs. Vet. Immunol. Immunopathol. 142, 95-636

100. 637

Nielsen, E.O., Nielsen, N.C., Friis, N.F., 2001. Mycoplasma hyosynoviae arthritis in grower-finisher 638

pigs. J. Vet. Med. A 48, 475-486. 639

Nielsen, E.O., Lauritsen, K.T., Friis, N.F., Enøe, C., Hagedorn-Olsen, T., Jungersen, G., 2005. Use 640

of a novel serum ELISA method and the tonsil-carrier state for evaluation of Mycoplasma 641

hyosynoviae distributions in pig herds with or without clinical arthritis. Vet. Microbiol. 111, 41-50. 642

Oh, Y., Seo, H.W., Han, K., Park, C., Chae, C., 2012. Protective effect of the maternally derived 643

porcine circovirus type 2 (PCV2)-specific cellular immune response in piglets by dam vaccination 644

against PCV2 challenge. J Gen Virol. 93, 1556-62. 645

Rautiainen, E., Wallgren, P., 2001. Aspects of the transmission of protection against Mycoplasma 646

hyopneumoniae from sow to offspring. J Vet Med B Infect Dis Vet Public Health. 48, 55-65. 647

- 33 -

Razin, S., Yogev, D., Naot, Y., 1998. Molecular biology and pathogenicity of mycoplasmas. 648

Microbiol. Mol. Biol. Rev. 62, 1094-1156. 649

Riber, U., Jungersen, G., 2007. Cell-mediated immune responses differentiate infections with 650

Brucella suis from Yersinia enterocolitica serotype O:9 in pigs. Vet. Immunol. Immunopathol. 116, 651

13-25. 652

Rooke, J.A., Bland, I.M., 2002. The acquisition of passive immunity in the new-born piglet. 653

Livestock Production Science 78, 13-23. 654

Ross, R.F., 1973. Predisposing factors in Mycoplasma hyosynoviae arthritis in swine. J. Infect. Dis. 655

127 (Supplement), S84-86. 656

Ross, R.F., Duncan, J.R., 1970. Mycoplasma hyosynoviae arthritis of swine. J. Am. Vet. Med. 657

Assoc. 157, 1515-1518. 658

Ross, R.F., Karmon, J.A., 1970. Heterogeneity among strains of Mycoplasma granularum and 659

identification of Mycoplasma hyosynoviae, sp. n. J. Bacteriol. 103, 707-713. 660

Ross, R.F., Spear, M.L., 1973. Role of the sow as a reservoir of infection for Mycoplasma 661

hyosynoviae. Am. J. Vet. Res. 34, 373-378. 662

Ross, R., Grebe, H., Kirchhoff, H., 1978. Serological and electrophoretic characteristics of 663

Mycoplasma hyosynoviae. Zentralbl. Veterinarmed. B 25, 444-451. 664

Salmon, H., 2000. Mammary gland immunology and neonate protection in pigs. Homing of 665

lymphocytes into the MG. Adv. Exp. Med. Biol. 480, 279-286. 666

Salmon, H., Berri, M., Gerdts, V., Meurens, F., 2009. Humoral and cellular factors of maternal 667

immunity in swine. Dev Comp Immunol. 33, 384-93. 668

- 34 -

Schollenberger, A., Degorski, A., Frymus, T., Schollenberger, A., 1986a. Cells of sow mammary 669

secretions. I. Morphology and differential counts during lactation. Zentralbl. Veterinarmed. A 33, 670

31-38. 671

Schollenberger, A., Frymus, T., Degorski, A., Schollenberger, A., 1986b. Cells of sow mammary 672

secretions. II. Characterization of lymphocyte populations. Zentralbl. Veterinarmed. A 33, 39-46. 673

Sibila, M., Bernal, R., Torrents, D., Riera, P., Llopart, D., Calsamiglia, M., Segalés, J., 2008. Effect 674

of sow vaccination against Mycoplasma hyopneumoniae on sow and piglet colonization and 675

seroconversion, and pig lung lesions at slaughter. Vet Microbiol. 127, 165-70. 676

Tuboly, S., Bernath, S., Glavits, R., Medveczky, I., 1988. Intestinal absorption of colostral 677

lymphoid cells in newborn piglets. Vet. Immunol. Immunopathol. 20, 75-85. 678

Wagstrom, E.A., Yoon, K.J., Zimmerman, J.J., 2000. Immune components in porcine mammary 679

secretions. Viral Immunol. 13, 383-397. 680

Wallgren, P., Bölske, G., Gustafsson, S., Mattsson, S., Fossum, C., 1998. Humoral immune 681

responses to Mycoplasma hyopneumoniae in sows and offspring following an outbreak of 682

mycoplasmosis. Vet Microbiol. 60, 193-205. 683

Williams, P.P., 1993. Immunomodulating effects of intestinal absorbed maternal colostral 684

leukocytes by neonatal pigs. Can. J. Vet. Res. 57, 1-8. 685

Yang, H., Oura, C.A., Kirkham, P.A., Parkhouse, R.M., 1996. Preparation of monoclonal anti-686

porcine CD3 antibodies and preliminary characterization of porcine T lymphocytes. Immunology. 687

88, 577-85. 688